Although the phosphorothioate (PT) modification, in which the nonbridging oxygen in the DNA sugar-phosphate backbone is replaced by sulfur, has been reported to play versatile roles in multiple cellular processes, very little data have been obtained to define the role of PT in epigenetic regulation. In this study, we report that the PT system in Salmonella enterica serovar Cerro 87 is involved in the transcriptional regulation of the torCAD operon encoding the trimethylamine N-oxide (TMAO) respiration machinery that enables the use of TMAO as a terminal electron acceptor for respiration when oxygen is not available. In vitro, PT enhanced the binding of the transcriptional activator of the torCAD operon, namely, TorR, to its DNA substrate (tor boxes). However, in vivo, the PT modification protein complex DndCDE downregulated torCAD transcription through competing with the binding of TorR to the tor boxes. The altered expression of torCAD caused by PT modification proteins affected cell growth that relied on TMAO respiration. To our knowledge, this is the first report supporting that PT proteins participate in transcriptional regulation, showing a new function of PT systems. IMPORTANCE Since the initial discovery of DNA phosphorothioate (PT) modification systems in Streptomyces lividans in the 1980s, explorations of the biological functions of DNA PT systems have advanced and yielded a number of important findings. However, the functions of PT systems, especially in genetic regulation, remain largely unknown. In this study, we report a case in which the PT system participates in the transcriptional regulation of the torCAD operon in Salmonella enterica serovar Cerro 87. While the PT modification enhanced the binding of TorR, the torCAD operon transcriptional activator, to its DNA substrate in vitro, we found that the PT modification protein complex DndCDE directly competed with TorR binding in vivo and subsequently repressed the expression of torCAD and attenuated cell growth that relied on TMAO respiration. These findings provide a deeper understanding of the characteristics of the PT chemical structure and broaden our understanding of the mechanisms by which PT regulates gene expression.
Keywords: DNA phosphorothioate modification; competitive binding; tor boxes.